Actin and tubulin play a central role in motility and the control of cell form. Given the crucial role of these proteins in both health and disease, the quaternary structures of actin and tubulin are important, since structural information can elucidate many questions of function. Specifically, this proposal is aimed at understanding the conformational changes that accompany actin polymerization and the hydrolysis of ATP, and the macromolecular organization of tubulin-tektin ribbons that appear to be key determinants of axonemal doublet microtubules. The main techniques that we will be used are electron microscopy and image analysis. An atomic structure for the actin monomer provides the basis for most of the actin research that is proposed. It is clear that the technique of combining x-ray and EM data will become an increasingly important tool in understanding the structure of complex macromolecular polymers and assemblies. Actin is a dynamic system, both in conformational changes that must occur within the protein during assembly as well as in terms of the ability of the assembled filament to undergo flexing and torsion. The techniques of electron microscopy and image analysis can be very useful in deriving dynamic information from static images. The application of these methods to actin should be able to shed light on the molecular motions that result from the hydrolysis of ATP after filament assembly, as well as on factors that appear to modulate the torsional and flexural rigidity of actin. However, electron microscopy cannot directly establish the time scales on which such motions occur, and collaborative spectroscopic studies to obtain this information are planned. While no crystal structure exists for tubulin, the proposed project on the tubulin-tektin ribbons, isolated from axonemes, stands on its own in the absence of high-resolution structures. The quaternary structure will be determined of these "ribbons", and such information will be of great relevance to understanding the assembly of the complicated axoneme which contains over 100 different proteins. Further, such studies may help explicate the structural organization of tektin within these ribbons, and provide a model for the folding of this protein family.